The methylotrophic yeast Pichia pastoris (syn. Komagataella sp.) is a well-established protein production host. Numerous strain engineering approaches for P. pastoris improved the productivity for various products and effort was also dedicated to promoters for production purposes (Prielhofer, R., M. Maurer, J. Klein, J. Wenger, C. Kiziak, B. Gasser & D. Mattanovich, (2013) Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Fact 12: 5). Gene promoters are key features for the expression of a gene of interest (GOI): transcription of RNA of a downstream (3′) GOI is driven by the upstream (5′) promoter sequence. RNA polymerase II (RNAPII) is responsible for transcription of mRNA in eukaryotes. RNAPII promoters consist of a core promoter and several cis-acting DNA elements: proximal promoter, enhancers, silencers and boundary/insulator elements. Yeast core promoters are typically located close (−75/+50 bp) to the main transcription initiation site, they frequently contain improper TATA boxes (up to 2 bases difference to the TATA consensus sequence) and lack promoter elements which are typically found in other organisms. Transcriptional regulation responds to different conditions and is conducted through by cis-acting elements and corresponding regulatory proteins (transcription factors (TFs)).
For biotechnological applications, promoters allowing either constitutive or regulated/inducible gene expression are used. Production processes utilizing P. pastoris favorably apply carbon source dependent promoters such as the methanol-inducible PAox. Thereby, the growth phase can be separated from the potentially burdening protein production phase. A set of promoters was recently reported (Prielhofer et al., 2013), which is also controlled by the carbon source, but does not rely on methanol for induction: These promoters share the feature of repression by excess glycerol and induction by limiting glucose. pG1 (SEQ ID 1), the strongest out of these promoters, is fully induced below 0.05 g/L glucose; it natively controls the expression of fa high-affinity glucose transporter gene GTH1. Glucose uptake characteristics are dependent on the presence of high and low affinity glucose transporters. Seventeen hexose transport (HXT) genes in S. cerevisiae (HXT1-17) are expressed depending on the glucose concentration, but only two HXT homologs are found in P. pastoris (PAS_chr1-4_0570 and PAS_chr2-1_0054, named PpHxt1 and PpHxt2). PpHxt1 was identified to be the major low-affinity transporter in P. pastoris, while high affinity glucose transport is facilitated by two other genes, namely PAS_chr3_0023 and PAS_chr1-3_0011 (GTH1, the gene controlled by pG1) Prielhofer et al., 2013).
While S. cerevisiae features a huge capacity of glucose uptake and (fermentative) glucose metabolism, P. pastoris has a lower glucose uptake rate and a respiratory metabolism of glucose. Furthermore, P. pastoris is able to take glucose at much lower extracellular concentrations than S. cerevisiae (KM of high-affinity transporters in the μM range in P. pastoris vs. mM range in S. cerevisiae). The fundamental difference in glucose uptake behavior is also displayed at the transcriptional control of related genes and can also be seen in the evolved functions of transcriptional regulators e. g. PpAft1 and PpMxr1 (homolog of ScAdr1).
P. pastoris promoter studies and random mutagenesis of PAOX1 and of the promoter of glyceraldehyde-3-phosphate dehydrogenase PGAP resulted in libraries with promoter variants possessing different activities, altered induction behavior compared to the wild-type promoter and in the identification of several important transcription factor binding sites (TFBS) of PAOX1 (WO2006/089329 A2).
The pG1 promoter and fragments thereof are further described in WO2013/050551 A1.
WO2014067926A1 discloses the expression of a protein of interest employing specific leader sequences. The leader were used with various promoter. As an exemplary promoter, the pG1 promoter is used.
Struhl K. (Proceedings of the National Academy of Sciences of the United States of America 1982, 78(7):4461-4465) describes deletion mapping of the yeast his3 promoter region. He concludes that the T-A-T-A box, a sequence in front of most eukaryotic genes is not sufficient for wild-type promoter function and suggests that the yeast promoter appears to be more complex than a simple site of interaction between RNA polymerase and DNA.
Quandt et al. (Nucleic Acids Research 1995, 23(23)4878-4884) describe tools for detection of consensus matches in nucleotide sequence data to identify regulatory motifs based on sequence data analysis. A library of consensus patterns was created and potential sequence matches were detected using a software tool (MatInspector).